Fusion PCR Protocol? - (Sep/22/2003 )

I am looking for a PCR Fusion protocol. (using pcr to make a fusion protein) Does anyone know where I can find one?Any help would be appreciated. Thanks

-bouttela-

QUOTE (bouttela @ Sep 22 2003, 04:21 AM)

I am looking for a PCR Fusion protocol. (using pcr to make a fusion protein) Does anyone know where I can find one?Any help would be appreciated. Thanks

I am trying to do the same. It worked for me with short sequences, but now is a headake. If you find something better, please, tell me!: Splicing by overlap extention by PCR.......A Warrens, M Jones and R. LechlerGene 186 (1997) 29-35and references therin

Then you PCR the 5' fragment with primers A and C and in a seperate tube PCR the 3' fragment using primers B and D. You do not need to get much of these fragments, so don't over PCR you'll just introduce mutations.

Load the samples on a clean gel And carefully cut out the two bands (LMP gel is best for this but a normal gel works fine also). Just cut out the most concentrated part in the gel no extra agarose. Add ~ 10X volume water, melt the agarose,mix and add ~3ul of each fragment solution to a new PCR mix (100ul total) (containing primes A and B only). Mix and place in PCR machine. heat to 94C 1', then cycle 94C 1' anneal 3' at atleast 5C below the overlap homology mt temperature extend longer then normal repeat 4X. Then PCR with correct conditions for primers A and B.

The gel part is importent to lose the primers and the original template.

-il0postino-

Disruption by Fusion PCRDavid Amberg and Ellen Beasley1) In separate PCR reactions, amplify the 5' and 3' ends of the gene of interest with primers about 200 bases apart. Primer 2 should begin with 24 nts complementary to the m13 forward primer(GTC GTG ACT GGG AAA ACC CTG GCG) and primer 3 should begin with 24 nts complementary to the m13 reverse primer (TCC TGT GTG AAA TTG TTA TCC GCT). PCR amplify the marker using the m13 forward and reverse primers. The Prakash and Jones vectors are useful for the marker PCR. The conditions for these PCR reactions are as follows:

6) Check for product on a mini gel. Phenol/chloroform extract the product and EtOH pptate and use directly to transform a diploid or gel purify the product and transform the diploid with this DNA.

Note: If PCR generated errors are of concern then each PCR generated fragment can be extracted from the low melt agarose and EtOH precipitated. Once free of the agarose, Vent should work in the fusion PCR reactions.